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Research Effects of different fibrinogen concentrations on blood loss and coagulation parameters in a pig model of coagulopathy with blunt liver injury Oliver Grottke*1,2, Till Braunschw

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Research

Effects of different fibrinogen concentrations on blood loss and coagulation parameters in a pig model of coagulopathy with blunt liver injury

Oliver Grottke*1,2, Till Braunschweig3, Dietrich Henzler4, Mark Coburn1, Rene Tolba2 and Rolf Rossaint1

Abstract

Introduction: The early application of fibrinogen could potentially reverse haemodilution-induced coagulopathy,

although the impact of varying concentrations of fibrinogen to reverse dilutional coagulopathy has not been studied

in vivo We postulated that fibrinogen concentration is correlated with blood loss in a pig model of coagulopathy with

blunt liver injury

Methods: Coagulopathy was induced in 18 anaesthetized pigs (32 ± 1.6 kg body weight) by replacing 80% of blood

volume with hydroxyethylstarch 130/0.4 and Ringer's lactated solution, and re-transfusion of erythrocytes Animals were randomly assigned to receive either 70 mg kg-1 (F-70) or 200 mg kg-1 (F-200) fibrinogen or placebo before

inducing blunt liver injury using a force of 225 ± 26 Newton Haemodynamics, coagulation parameters and blood loss were monitored for 2 hours After death, histological examination of internal organs was performed to assess the presence of emboli and the equality of liver injury

Results: Plasma dilution caused severe coagulopathy Measured by thromboelastography fibrinogen restored

coagulation dose-dependently Total blood loss was significantly lower and survival better in both fibrinogen groups as

compared to controls (P < 0.05) Between the F-70 (1317 ± 113 ml) and the F-200 group (1155 ± 232 ml) no significant difference in total blood loss could be observed, despite improved coagulation parameters in the F-200 group (P <

0.05) Microscopy revealed even injury pattern and no (micro) thrombi for either group

Conclusions: Restoring fibrinogen with 70 or 200 mg kg-1 after severe dilutional coagulopathy safely improved

coagulation and attenuated blood loss after experimental blunt liver trauma The higher dosage of fibrinogen was not associated with a further reduction in blood loss

Introduction

Traumatised and surgical patients with massive

haemor-rhage are predisposed to develop coagulopathy, as a

result of multiple mechanisms including acidosis,

hypo-thermia, anaemia, hyperfibrinolysis and

hypotension-induced inflammation, as well as consumption and

dilu-tion of coaguladilu-tion factors [1] Diludilu-tional coagulopathy

may occur after massive blood loss, as crystalloid and

col-loid solutions are infused for fluid resuscitation The

degree of coagulopathy depends on the type and volume

of the fluids infused [2] Resuscitation with colloid

plasma expanders may lead to a functional fibrinogen

deficiency by abnormal fibrin polymerisation, which can

be reversed by exogenous fibrinogen [3] Furthermore, fibrinogen concentrations of less than 100 mg dL-1 may occur before other coagulation factors are diluted [4] The early decrease of fibrinogen levels has led to the hypothesis that fibrinogen is a key factor for reversing haemodilution induced coagulopathy Fresh frozen plasma (FFP), cryoprecipitate and fibrinogen may be sub-stituted to restore low concentrations of fibrinogen FFP contains all coagulation factors and is recommended according to international guidelines to be used either in massive bleeding or if a prolongation of prothrombin time (PT) activated thromboplastin time of more than 1.5 times is accompanied by signs of microvascular bleeding [5] However, to restore coagulation with FFP and

* Correspondence: ogrottke@ukaachen.de

1 Department of Anaesthesiology, RWTH Aachen University Hospital

Pauwelsstrasse 30, D-52074 Aachen, Germany

Full list of author information is available at the end of the article

increase low levels of fibrinogen efficiently, large volumes

of FFP are needed Other drawbacks of FFP transfusion

include immunological reactions such as

transfusion-related lung injury, anaphylaxis and haemolysis in cases

of ABO incompatibility [6] Alternatively, cryoprecipitate

might be used to raise critical levels of fibrinogen,

con-taining factor VIII, fibrinogen, fibronectin, von

Wille-brand factor and factor XIII [7] A dose of around 10

single bags of cryoprecipitate derived from units of whole

blood typically raises the plasma fibrinogen level by up to

60 to 100 mg dL-1 However, due to the risk of

blood-borne pathogen transmission, the use of cryoprecipitate

for this indication is discussed critically In respect to FFP

and cryoprecipitate pasteurised fibrinogen is virus

inacti-vated In a pioneering study Fries and colleagues could

demonstrate that the early application of 250 mg kg-1

fibrinogen reversed haemodilution-induced

coagulopa-thy and reduced blood loss in a porcine model of liver

injury [8] Subsequent studies confirmed these findings

and also showed a dose-dependent effect of fibrinogen

substitution on thromboelastometry variables in vitro

[9-11] However, as in vitro studies are performed under low

shear conditions, the effects of the interaction with

vas-cular endothelium were not investigated and relevant

clinical outcomes in relation to increasing doses of

fibrin-ogen, such as blood loss and survival rate, remained

unknown [12,13] Therefore this study investigated a

pos-sible dose-dependent effect of fibrinogen to reverse

hae-modilution in vivo in a model of blunt liver injury.

Primary endpoints of this study included blood loss and

survival time, secondary endpoints were improvement in

coagulation tests including thromboelastometry and the

evaluation of adverse events

Materials and methods

Ethics and anaesthesia

All experiments were performed in accordance with the

German legislation governing animal studies following

The Principles of Laboratory Animal Care [14] Official

permission for this study was granted from the

govern-mental animal care and use office (Landesamt für Natur,

Umwelt und Verbraucherschutz Nordrhein-Westfalen,

Recklinghausen, Germany)

Before surgery, pigs were housed in ventilated rooms

and allowed to acclimatise to their surroundings for a

minimum of five days Animals were fasted overnight

before surgical procedure, with water allowed ad libitum.

Eighteen German male land-race pigs, weighing (mean

± standard deviation (SD)) 32 ± 1.6 kg received an

intra-muscular injection of 4 mg kg-1 azaperone (Stresnil™,

Janssen, Neuss, Germany) as pre-medication

Anaesthe-sia was induced by an intravenous injection of 3 mg kg-1

propofol (Disoprivan®, Astra Zeneca, Wedel, Germany)

followed by orotracheal intubation The animals were

ventilated with 20 to 26 breaths min-1 and a tidal volume

of 10 mL kg-1 to keep the end-tidal partial pressure of car-bon dioxide (pCO2) between 36 and 42 mmHg The inspiratory oxygen fraction was 1.0 during haemodilution and reduced to 0.4 afterwards Anaesthesia was main-tained with isoflurane at end-tidal concentrations of 1%

to 1.2% and continuous infusion of fentanyl at 3 μg kg-1 h

-1 Ringer's lactated solution (RL) was infused at 4 mL kg

-1h-1 at first and increased to 8 mL kg-1h-1after laparotomy until infliction of trauma Body temperature was main-tained over the entire experiment (36.5 to 37.0°C) with a warming blanket

Monitoring included electrocardiography, tail pulse oximetry, temperature and arterial and central venous pressure by femorally introduced catheters connected to

a standard anaesthesia monitor (AS/3, Datex Ohmeda, Helsinki, Finland)

Surgical preparation and haemodilution

Two 8.5 Fr catheters were surgically implanted in the right and left jugular veins for volume substitution and insertion of a pulmonary artery catheter A splenectomy was performed under neuromuscular blockage with pan-curonium (0.2 mg kg-1 intravenous) To compensate for blood loss associated with the splenectomy, a bolus of warmed RL three times the weight of the spleen was administered To achieve comparable low concentrations

of fibrinogen, intravascular volume was diluted by replac-ing approximately 80% of the estimated blood volume [15] by hydroxyethylstarch 130/0.4 (Voluven®, Fresenius, Bad Homburg, Germany) with a maximum dose of 50 ml

kg-1 and RL in a ratio of 1:1.2 to 1.5 The collected blood was processed (Cell Saver 5®, Haemonetics, Munich, Ger-many) and the red cells were re-transfused before trauma infliction to avoid early death from severe anaemia

Fibrinogen substitution and liver injury

Six animals each were randomised to receive normal saline solution (controls), 70 mg kg-1 fibrinogen (group F-70; fibrinogen: Haemocompletan®, CSL Behring, Mar-burg, Germany), or 200 mg kg-1 (group F-200) fibrinogen Subsequently, a grade III blunt liver injury [16] was inflicted as described before using a custom-made instru-ment [17] Briefly, the liver was gently retracted to allow adequate exposure The base of the plate was positioned beneath the right middle lobe The injury was induced by one-time clamping of the instrument through the paren-chyma with a force of 225 ± 26 Newton (N) The force of injury was analysed in real-time and the signal was dis-played in a visual programming environment (LabView 8.8, National Instruments, Austin, TX, USA) after ampli-fying (VG140, ATR Industrie-Elektronik, Krefeld, Ger-many) and digitizing the force signal (NI USB-6009, National Instruments, Austin, TX, USA) The time of

reg-istration was set to 500 msec In all cases the injury was

inflicted by the same investigator (OG), being also

blinded to the experimental group

After liver injury, the abdomen was closed with staples

and further manipulations were avoided Five minutes

after injury all animals received 4 mL kg -1 min-1 of RL

given over eight minute Afterwards, the rate was set to

25 mL kg-1 h-1 until the end of the experiment The

obser-vation period ended at 120 minutes after injury Pulseless

electrical activity, a mean arterial pressure of less than 10

mmHg and an end-tidal PCO2 of less than 10 mmHg

were defined as death Animals surviving for more than

two hours were killed with fentanyl, propofol and

potas-sium chloride Immediately after death, the abdomen was

reopened, the vena cava was clamped cranial to the liver

and the intraperitoneal blood was collected to determine

the total blood loss post-injury After that, internal organs

(lungs, heart, liver and kidneys) were removed and

pre-pared for histological examination

Blood sampling and analytical methods

Blood was collected and arterial blood gas analysis were

performed 10 minutes after splenectomy ('baseline'), at

the end of haemodilution ('haemodilution'), after

fibrino-gen substitution ('fibrinofibrino-gen') and 120 minutes after liver

injury ('trauma') or immediately following death,

which-ever occurred first Haemoglobin concentration, pH

value, partial pressure of oxygen (pO2) and carbon

diox-ide (pCO2) were measured with a blood gas analyser

(ABL500, Radiometer, Copenhagen, Denmark)

Pro-thrombin time (PT), activated partial thromboplastin

time (aPTT) and fibrinogen concentrations were

deter-mined by standard laboratory methods using the

appro-priate tests from Dade Behring (Marburg, Germany) on a

coagulometer (KC4, Baxter, Newbury, UK)

Thrombin-antithrombin (TAT) complexes were quantified by ELISA

(TAT, Dade Behring, Germany) A coagulation analyser

(ROTEM®, Pentapharm, Munich, Germany) was used for

thrombelastometry with the EXTEM® assay according to

the manufacturer's instructions The following

parame-ters were obtained: clot formation time (CFT in seconds:

reflects the coagulation time until 20 mm of amplitude

are reached), maximum clot firmness (MCF in mm:

reflects the strength of a resulting clot) and the α-angle

(in degree: shows the rate of fibrin polymerisation)

Pathological examination

All investigated internal organs, such as the lungs, the

heart, liver and the kidneys, were removed after death

and directly fixed in 10% buffered formalin Injured parts

of the liver were cut into 3 mm thick slices Only areas of

maximum depth of injury and most severe vessel rupture

were chosen for further histological examination In

addi-tion representative tissue secaddi-tions of all four organs were

processed to explore for thrombotic events All samples were embedded in paraffin and stained by H&E and a standard Elastica-van Gieson protocol for histological examination under light microscopy (Eclipse 50i, Nikon, Duesseldorf, Germany) Secondary, suspect sections of lung and liver tissues were immunostained for fibrinogen and von Willebrand factor A polyclonal rabbit antihu-man fibrinogen antibody (DAKO A0080, polyclonal rab-bit, DAKO, Glostrup, Denmark) and polyclonal rabbit von Willebrand factor VIII antibody (DAKO, A0082, polyclonal rabbit) were used at a concentration of 1:100 For staining, the ABC Vectastain universal kit (Vector Laboratories, Burlingame, CA, USA) and haematoxylin as counterstain was used A blinded pathologist subse-quently assessed the degree of injury in the liver and of fibrin deposition in vessels and microthrombi formation

in all organs

Statistical analysis

Data are presented as mean ± SD (SPSS V16, Chicago, IL, USA) Normal distribution of parameters was shown on the interpretation of Q-Q plots and histograms Differ-ences between groups were analysed with a one-way

analysis of variance (ANOVA) with Scheffe's post hoc test

and Games Howell for multiple comparisons, respec-tively A repeated measures ANOVA was applied to anal-yse the influence of dilution and treatment substitution

over time using Scheffe's post hoc and Games Howell

tests

Non-parametric distributed parameters of throm-boelastometry were analysed using Kruskal-Wallis H-test and Bonferroni-Dunn tests for multiple comparisons Data are presented in box plots Data on survival were analysed by the log-rank test Statistical tests were per-formed two-tailed and the level of significance was

defined as P < 0.05.

Results Baseline measurements and coagulation parameters after haemodilution

Baseline parameters were comparable between groups (Tables 1 and 2) The dilution caused a significant coagul-opathy and a drop in platelets PT increased from 9.3 ± 0.7 seconds to 19 ± 2 seconds (pooled data) whereas fibrinogen concentrations decreased from 301 ± 36 mg

dL-1 to 54 ± 7 mg dL-1 (P < 0.001; Figure 1) Coagulopathy

was adequately detected by significant findings in

throm-boelastometry (P < 0.001; Figure 2) No clinical signs of

coagulopathy, such as oozing from insertion sites or mucosal bleeding, were observed in any group

Coagulation parameters after fibrinogen substitution and after injury

Fibrinogen substitution significantly increased the con-centrations of fibrinogen in the intervention groups

(F-70: 148 ± 7 mg dL-1; F-200: 237 ± 17 mg dL-1) Although

PT decreased equally in both intervention groups (Figure

1), the decrease in CFT and the increases in MCF and

α-angle were dose dependent (Figure 2)

After haemodilution and liver injury fibrinogen

con-centrations decreased in all groups over time However,

in the F-200 group, fibrinogen concentration was

signifi-cantly higher (131 ± 26 mg dL-1) and PT lower (11 ± 1

seconds) than in F-70 group (fibrinogen: 67 ± 11 mg dL-1,

PT 17 ± 2 seconds) Corresponding results were obtained

by thromboelastometry (Figure 2), with controls showing

consistently lowest (MCF, α-angle), respectively highest

values (CFT)

The aPTT was significantly prolonged after dilution,

without significant differences between groups Similarly,

TAT complexes increased in all groups without

signifi-cant differences between groups (Table 1)

Concentra-tions of D-Dimer were below 500 μg l-1 at all times (data

not shown)

Haemodynamics and blood loss

No differences in haemodynamics were observed

between groups until liver injury (Table 2) Following

liver injury all animals developed haemorrhagic shock

Blood loss after liver injury was highest in the control

group (1803 ± 248 ml; P < 0.05), followed by the F-70

(1317 ± 113 ml) and F-200 (1155 ± 232 ml) groups The

difference in blood loss between the intervention groups

was not significant (P = 0.205) Mean arterial pressure

and cardiac output were significantly lower in controls than in the intervention groups

All animals in the control group died before the end of the observation period, with a survival time of 59 ± 12 minutes (Figure 3) All animals in the F-200 group sur-vived, whereas two out of six animals (33%) of the F-70

group died before the end of the observation time (P =

0.138)

Histopathological analysis

Macroscopical and histological evaluation by immunos-taining with von Willebrand factor of injured liver sec-tions revealed an equal tissue damage as well as comparable laceration of venous vessels of a maximum of

3 to 4 mm No evidence for thrombus formation or microthrombi was found in the H&E and fibrinogen stain

of kidney, heart or lung tissues

Discussion

In this in vivo study we could demonstrate a

dose-depen-dent effect of fibrinogen to reverse haemodilution-induced coagulopathy Increasing concentrations of fibrinogen resulted in a further improvement of clot for-mation and clot firmness Blood loss after liver injury was significantly lower in the fibrinogen groups as compared

Table 1: Laboratory parameters (mean ± standard deviation) Parameters included in the table are haemoglobin, platelet count (PLT), prothrombin time (PT), activated partial thromboplastin time (aPTT) and thrombin-AT complex (TAT) at baseline, after haemodilution, after fibrinogen substitution (fibrinogen) at the end of the observation period (trauma)

Haemoglobin (g L -1 )

PLT (10 3 μL -1 )

aPTT (s)

TAT (μg L -1 )

*P < 0.005 F-200 vs control.

with controls, but there was no difference between

sub-stituting 70 or 200 mg kg-1 fibrinogen in regards to blood

loss or survival

After haemodilution, coagulation was severely

impaired as shown by prolonged PT, clot formation and

an overall reduction in clot firmness To meet a plasma

fibrinogen concentration below the threshold of

interna-tional recommendations (> 80 to 100 mg dL-1) [5], the

degree of haemodilution was set to achieve a fibrinogen

concentration of approximately 50 mg dL-1 Although it is

well known that colloids may interfere with

concentra-tions of fibrinogen determined by the Clauss method

[18], the prolongation of clot formation and decreased

clot strength confirmed the haemodilution induced

coag-ulopathy in our study

In concordance with several in vitro studies we could

show that increasing concentrations of fibrinogen dose

dependently improved clot formation (lower CFT and

higher α-angle) and clot strength (increase of MCF) using

the EXTEM® assay [9-11] Although the FIBTEM® assay

specifically attributes the impact of fibrinogen/fibrin on clot strength by inhibiting platelets through

cytochalasin-D [19], the FIBTEM® assay cannot be reliable used with porcine blood [20] The observed improvement on clot strength after fibrinogen substitution is most likely explained by its binding to GIIb/IIIa receptors, as the platelet count did not significantly vary after haemodilu-tion and fibrinogen substituhaemodilu-tion The abundant number

of approximately 40,000 to 50,000 GIIb/IIIa receptors per activated platelet allows binding of large amounts of fibrinogen [21] This theory is supported by a possible compensating role of fibrinogen with the presence of thrombocytopenia [22,23] However, even at very high doses of exogenous fibrinogen, clot strength did not reach baseline values, probably because of reduced levels

of other coagulation factors such as FXIII [24-26] In con-trast, prolonged PTs were reversed after fibrinogen provi-sion, but this effect was not dose dependent As the PT only reflects 5% of the whole coagulation process the sub-stitution of 70 mg kg-1 of fibrinogen already normalised

Table 2: Haemodynamic parameters (mean ± standard deviation) Parameters included in the table are heart rate (HR), mean arterial pressure (MAP), central venous pressure (CVP), mean pulmonary pressure (MPAP) and cardiac output (CO) at baseline (after splenectomy), after fibrinogen substitution (fibrinogen), haemodilution, five minutes after trauma and at the end of the observation period (trauma)

Baseline Haemodilution Fibrinogen 5 min after

Trauma

Trauma

HR (beats min -1 )

MAP (mmHg)

CVP (mmHg)

MPAP (mmHg)

CO (L min -1 )

*P < 0.005 vs control.

PT down to almost baseline values despite altered

throm-boelastometry variables [27] Our observation confirms

that the sensitivity of PT is insufficient to guide

haemo-static therapy in haemodilution-induced coagulopathy

[28] A recommendation by the Society of Thoracic

Sur-geons therefore suggests guiding haemostatic therapy by

point-of-care testing rather than by plasma-based

coagu-lation assays [29]

Following trauma, haemodilution and shock decreased

both the clot formation and clot strength in all animals

This effect is due to the loss, consumption and dilution of

coagulation factors However, at the end of the

observa-tion period clot strength and fibrinogen concentraobserva-tion

were still higher in the F-200 group as compared with the

F-70 group, but not associated with a further reduction in

blood loss Thus the endogenous potential of

procoagu-lant activators was sufficient to activate fibrinogen and to

terminate bleeding The comparable blood loss between

the F-70 and F-200 group and the similar decrease of the

mean concentration of fibrinogen indicates that the

limit-ing factor determinlimit-ing the time to haemostasis was rather

restricted by the concentration of fibrinogen than its

acti-vation Although thrombin generation and clot formation

have been shown to be decreased at a plasma dilution of

more than 40% [30], the residual thrombin is usually

suf-ficient to cleave fibrinogen However, trauma-induced

blood loss and haemodilution further reduced the rate of

thrombin generation that is crucial to achieve sufficient

haemostasis Due to the short half-life time of FXa and

thrombin [31] clinical situations with dilutional

coagul-opathy and active bleeding might also require the

addi-tional substitution of procoagulant factors [32]

Although some recent studies indicate a potential pro-tective effect of higher levels of fibrinogen to reduce blood loss [33-36], fibrinogen is rarely used as monother-apy but as an adjunct in clinical situations with life-threatening bleeding Thus the substitution of fibrinogen may be a reasonable approach to reduce the use of allo-genic blood products, but the efficacy of fibrinogen sub-stitution may also be enhanced by the concomitant application of other coagulation factors

There is some concern that substitution of fibrinogen may enhance the risk for thromboembolic events This risk may be aggravated by the concomitant application of other haemostatics, such as antifibrinolytics Although some studies indicate an association between chronic ele-vation of fibrinogen and an increased risk for cardiovas-cular events [37], a systematic review about the safety of fibrinogen substitution in a situation of deficit showed a low thrombogeneity [38] Our results are consistent with these studies, as we could not detect (micro) vascular thrombosis or hypercoagulability after fibrinogen substi-tution

Some limitations do apply Despite the application of

up to 200 mg kg-1 fibrinogen, thromboelastometry vari-ables were not restored to baseline values The provision

of other coagulation factors, such as FXIII might have shown different results The experimental setup required inducing the haemodilution and fibrinogen application before the infliction of trauma It does not exactly mirror

a clinical situation, where coagulopathy occurs after trauma Although we mimicked a blunt liver injury, we allowed free bleeding after the injury, which is somewhat similar to a clinical situation with penetrating trauma

Figure 1 Prothrombin time and fibrinogen concentrations at baseline, after haemodilution, fibrinogen substitution (fibrinogen) and

trau-ma Data presented as mean ± standard deviation.















 

 








 


 


 


















   

Further, the observation time of this study was limited to only two hours, which prevented the study of further treatment effects or possible physiological compensation mechanisms However, all of the control animals had died within this observation period, which demonstrates a clear treatment effect In addition, the induction of injury was performed in anaesthetised healthy pigs Thus, the physiological response to such things as pain and inflam-mation may have additional effects on haemostasis, which are not reflected in our model

Finally, there is a great debate about the ideal resuscita-tion fluid, which has been recently addressed in a Cochrane systematic review [39] It would be way beyond the scope of this study to discuss the implications of the fluids needed to maintain haemodynamic stability in our pig model of severe dilutional coagulopathy Both crystal-loid and colcrystal-loid solutions have manifold influence on the coagulation system, inflammatory responses and organ function Our model of a combination of hydroxyethyl-starch and crystalloid represents the current concept at our institution, and probably of many other centres, for resuscitation of haemorrhagic shock, until the optimum resuscitation strategy has been identified

Conclusions

In summary, dilutional coagulopathy could be reversed

by the early administration of exogenous fibrinogen in the absence of severe anaemia Higher doses of fibrino-gen correlated with improved parameters of throm-boelastometry and may be a reasonable approach to reduce the use of FFP, platelet concentrate and red blood cells as these allogenic blood products are associated with various adverse outcomes However, the results of our study also show that substituting fibrinogen concentra-tion to values of more than 150 mg dL-1 had no additional effect on clinical relevant endpoints in this specific

ani-Figure 2 Thromboelastometry parameters (a) Clot formation time,

(b) Maximum clot firmness and (c) α-angle at various time points

in-cluding baseline, after haemodilution, fibrinogen substitution

(fibrino-gen) and trauma Results are shown as box plots (minimum, first

quartile, median, third quartile, maximum) *P < 0.05 vs control; †P <

0.05 vs F-70.



A





Trauma Fibrinogen

Haemodilution Baseline

80

70

60

50

40

30

20

F-200 F-70

Control

gr

Trauma Fibrinogen

Haemodilution Baseline

90

80

70

60

50

40

30

F-200 F-70

Control

gr

B

C



















gr


























Figure 3 Data of survival are presented as a Kaplan-Meier curve

*P < 0.05 vs control.



















mal model, if no other coagulation factors or

thrombo-cytes were transfused Thus, future clinical studies should

address the question of optimum level of fibrinogen in

combination with the replacement of other clotting

fac-tors, timing of fibrinogen substitution and patient

selec-tion

Key messages

• We could demonstrate that restoring fibrinogen

with 70 or 200 mg kg-1 after severe dilutional

coagul-opathy dose dependently improved coagulation

parameters as shown by thromboelastometry

vari-ables

• Although blood loss after liver injury was

signifi-cantly lower in the fibrinogen groups as compared

with controls, there was no difference between

substi-tuting 70 or 200 mg kg-1 fibrinogen in regard to blood

loss or survival Therefore substituting fibrinogen

concentration to values above 150 mg dL-1 had no

additional effect on clinical relevant endpoints in this

specific animal model

• As no (micro) vascular thrombosis or

hypercoagula-bility was observed, the early application of fibrinogen

might be a safe approach to restore critical

concentra-tions of fibrinogen and may reduce the need for the

transfusion of allogenic blood products

Abbreviations

ANOVA: analysis of variance; aPTT: activated partial thromboplastin time; CFT:

clot formation time; ELISA: enzyme-linked immunosorbent assay; FFP: fresh

frozen plasma; H&E: haematoxylin and eosin; MCF: maximum clot firmness;

pCO2: partial pressure of carbon dioxide; pO2: partial pressure of oxygen; PT:

prothrombin time; RL: Ringer's lactated solution; SD: standard deviation; TAT:

thrombin-antithrombin.

Competing interests

RR has received honoraria for lectures and consultancy from CSL Behring,

Ger-many Fibrinogen (Haemocompletan ® ) was provided by CSL Behring, Marburg,

Germany for the current study, but there was not financial support The

authors declare that they have no other competing interests.

Authors' contributions

OG conceived and conducted the experimental laboratory work, performed

the statistical analysis and drafted the manuscript RR participated in the study

design and coordination and helped to draft the manuscript TB, MC, and RT

helped to perform the study and draft the manuscript DH draft the

manu-script All authors read and approved the final manumanu-script.

Acknowledgements

The authors thank Thaddäus Stopinski, Dr Kira Scherer (Institute of Laboratory

Animal Science), Daniela Smeets and Eduardo Lee (Institute of Pathology) for

their excellent support.

Author Details

1 Department of Anaesthesiology, RWTH Aachen University Hospital

Pauwelsstrasse 30, D-52074 Aachen, Germany, 2 Institute for Laboratory Animal

Science, RWTH Aachen University Hospital, Pauwelsstrasse 30, D-52074

Aachen, Germany, 3 Department of Pathology, RWTH Aachen University

Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany and 4 Department of

Anaesthesia and Division of Critical Care, Dalhousie University Halifax, Queen

Elisabeth II Health Sciences Center, 10 West Victoria, 1276 South Park St.,

Halifax, NS, B3H 2Y9, Canada

References

1 Hess JR, Brohi K, Dutton RP, Hauser CJ, Holcomb JB, Kluger Y, Mackway-Jones K, Parr MJ, Rizoli SB, Yukioka T, Hoyt DB, Bouillon B: The

coagulopathy of trauma: a review of mechanisms J Trauma 2008,

65:748-754.

2 Mittermayr M, Streif W, Haas T, Fries D, Velik-Salchner C, Klingler A, Innerhofer P: Hemostatic changes after crystalloid or colloid fluid administration during major orthopedic surgery: the role of fibrinogen

administration Anesth Analg 2007, 105:905-917.

3 Fenger-Eriksen C, Anker-Møller E, Heslop J, Ingerslev J, Sørensen B: Thrombelastographic whole blood clot formation after ex vivo addition of plasma substitutes: improvements of the induced

coagulopathy with fibrinogen concentrate Br J Anaesth 2005,

94:324-329.

4 Hiippala ST, Myllylä GJ, Vahtera EM: Hemostatic factors and replacement

of major blood loss with plasma-poor red cell concentrates Anesth

Analg 1995, 81:360-365.

5 Spahn DR, Cerny V, Coats TJ, Duranteau J, Fernández-Mondéjar E, Gordini

G, Stahel PF, Hunt BJ, Komadina R, Neugebauer E, Ozier Y, Riddez L, Schultz A, Vincent JL, Rossaint R, Task Force for Advanced Bleeding Care in Trauma: Management of bleeding following major trauma: a European

guideline Crit Care 2007, 11:R17.

6 O'Shaughnessy DF, Atterbury C, Bolton Maggs P, Murphy M, Thomas D, Yates S, Williamson LM, British Committee for Standards in Haematology, Blood Transfusion Task Force: Guidelines for the use of fresh-frozen

plasma, cryoprecipitate and cryosupernatant Br J Haematol 2004,

126:11-28.

7 Callum JL, Karkouti K, Lin Y: Cryoprecipitate: the current state of

knowledge Transfus Med Rev 2009, 23:177-188.

8 Fries D, Krismer A, Klingler A, Streif W, Klima G, Wenzel V, Haas T, Innerhofer P: Effect of fibrinogen on reversal of dilutional

coagulopathy: a porcine model Br J Anaesth 2005, 95:172-177.

9 Fries D, Innerhofer P, Reif C, Streif W, Klingler A, Schobersberger W, Velik-Salchner C, Friesenecker B: The effect of fibrinogen substitution on

reversal of dilutional coagulopathy: an in vitro model Anesth Analg

2006, 102:347-351.

10 Haas T, Fries D, Velik-Salchner C, Reif C, Klingler A, Innerhofer P: The in vitro effects of fibrinogen concentrate, factor XIII and fresh frozen

plasma on impaired clot formation after 60% dilution Anesth Analg

2008, 106:1360-1365.

11 Bolliger D, Szlam F, Molinaro RJ, Rahe-Meyer N, Levy JH, Tanaka KA: Finding the optimal concentration range for fibrinogen replacement

after severe haemodilution: an in vitro model Br J Anaesth 2009,

102:793-799.

12 Ruggeri ZM: The role of von Willebrand factor and fibrinogen in the

initiation of platelet adhesion to thrombogenic surfaces Thromb

Haemost 1995, 74:460-463.

13 Ruggeri ZM: Mechanisms initiating platelet thrombus formation

Thromb Haemost 1997, 78:611-616.

14 Guide for the care and use of laboratory animals In Institute of

Laboratory Animal Resources, Commission on Life Sciences, National Research Council 7th edition Washington DC: National Academy Press;

1996

15 Bush JA, Jesen WN, Cartwright GE, Wintrobe MM: Blood volume studies

in normal and anemic swine Am J Physiol 1955, 181:9-14.

16 Moore EE, Cogbill TH, Jurkovich GJ, Shackford SR, Malangoni MA,

Champion HR: Organ injury scaling: spleen and liver (1994 revision) J

Trauma 1995, 38:323-324.

17 Grottke O, Braunschweig T, Philippen B, Gatzweiler KH, Gronloh N, Staat

M, Rossaint R, Tolba R: A new model for blunt liver injuries in the swine

Eur Surg Res 2010, 44:65-73.

18 Hiippala ST: Dextran and hydroxyethyl starch interfere with fibrinogen

assays Blood Coagul Fibrinolysis 1995, 6:743-746.

19 Lang T, Toller W, Gütl M, Mahla E, Metzler H, Rehak P, März W, Halwachs-Baumann G: Different effects of abciximab and cytochalasin D on clot

strength in thrombelastography J Thromb Haemost 2004, 2:147-153.

20 Velik-Salchner C, Schnürer C, Fries D, Müssigang PR, Moser PL, Streif W, Kolbitsch C, Lorenz IH: Normal values for thrombelastography (ROTEM)

Received: 24 January 2010 Revised: 9 March 2010 Accepted: 14 April 2010 Published: 14 April 2010

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Critical Care 2010, 14:R62

and selected coagulation parameters in porcine blood Thromb Res

2006, 117:597-602.

21 Wagner CL, Mascelli MA, Neblock DS, Weisman HF, Coller BS, Jordan RE:

Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding

to human platelets Blood 1996, 88:907-914.

22 Velik-Salchner C, Haas T, Innerhofer P, Streif W, Nussbaumer W, Klingler A,

Klima G, Martinowitz U, Fries D: The effect of fibrinogen concentrate on

thrombocytopenia J Thromb Haemost 2007, 5:1019-1025.

23 Lang T, Johanning K, Metzler H, Piepenbrock S, Solomon C, Rahe-Meyer N,

Tanaka KA: The effects of fibrinogen levels on thromboelastometric

variables in the presence of thrombocytopenia Anesth Analg 2009,

108:751-758.

24 Nielsen VG, Gurley WQ Jr, Burch TM: The impact of factor XIII on

coagulation kinetics and clot strength determined by

thrombelastography Anesth Analg 2004, 99:120-123.

25 Schroeder V, Chatterjee T, Kohler HP: Influence of blood coagulation

factor XIII and FXIII Val34Leu on plasma clot formation measured by

thrombelastography Thromb Res 2001, 104:467-474.

26 Nielsen VG, Cohen BM, Cohen E: Effects of coagulation factor deficiency

on plasma coagulation kinetics determined via thrombelastography:

critical roles of fibrinogen and factors II, VII, X and XII Acta Anaesthesiol

Scand 2005, 49:222-231.

27 Negrier C, Dargaud Y, Bordet JC: Basic aspects of bypassing agents

Haemophilia 2006, 12:48-52.

28 Kheirabadi BS, Crissey JM, Deguzman R, Holcomb JB: In vivo bleeding

time and in vitro thrombelastography measurements are better

indicators of dilutional hypothermic coagulopathy than prothrombin

time J Trauma 2007, 62:1352-1359.

29 Society of Thoracic Surgeons Blood Conservation Guideline Task Force,

Ferraris VA, Ferraris SP, Saha SP, Hessel EA, Haan CK, Royston BD, Bridges

CR, Higgins RS, Despotis G, Brown JR, Society of Cardiovascular

Anesthesiologists Special Task Force on Blood Transfusion, Spiess BD,

Shore-Lesserson L, Stafford-Smith M, Mazer CD, Bennett-Guerrero E, Hill

SE, Body S: Perioperative blood transfusion and blood conservation in

cardiac surgery: the Society of Thoracic Surgeons and The Society of

Cardiovascular Anesthesiologists clinical practice guideline Ann

Thorac Surg 2007, 83:S27-S86.

30 Schols SE, Feijge MA, Lancé MD, Hamulyák K, ten Cate H, Heemskerk JW,

van Pampus EC: Effects of plasma dilution on tissue-factor-induced

thrombin generation and thromboelastography: partly compensating

role of platelets Transfusion 2008, 48:2384-2394.

31 Jesty J, Beltrami E: Positive feedbacks of coagulation: their role in

threshold regulation Arterioscler Thromb Vasc Biol 2005, 25:2463-2469.

32 Schols SE, Meijden PE van der, van Oerle R, Curvers J, Heemskerk JW, van

Pampus EC: Increased thrombin generation and fibrinogen level after

therapeutic plasma transfusion: relation to bleeding Thromb Haemost

2008, 99:64-70.

33 Charbit B, Mandelbrot L, Samain E, Baron G, Haddaoui B, Keita H, Sibony O,

Mahieu-Caputo D, Hurtaud-Roux MF, Huisse MG, Denninger MH, de Prost

D, PPH Study Group: The decrease of fibrinogen is an early predictor of

the severity of postpartum hemorrhage J Thromb Haemost 2007,

5:266-273.

34 Karlsson M, Ternström L, Hyllner M, Baghaei F, Flinck A, Skrtic S, Jeppsson

A: Prophylactic fibrinogen infusion reduces bleeding after coronary

artery bypass surgery A prospective randomised pilot study Thromb

Haemost 2009, 102:137-144.

35 Rahe-Meyer N, Pichlmaier M, Haverich A, Solomon C, Winterhalter M,

Piepenbrock S, Tanaka KA: Bleeding management with fibrinogen

concentrate targeting a high-normal plasma fibrinogen level: a pilot

study Br J Anaesth 2009, 102:785-792.

36 Fenger-Eriksen C, Jensen TM, Kristensen BS, Jensen KM, Tønnesen E,

Ingerslev J, Sørensen B: Fibrinogen substitution improves whole blood

clot firmness after dilution with hydroxyethyl starch in bleeding

patients undergoing radical cystectomy: a randomized,

placebo-controlled clinical trial J Thromb Haemost 2009, 7:795-802.

37 Tatli E, Ozcelik F, Aktoz M: Plasma fibrinogen level may predict critical

coronary artery stenosis in young adults with myocardial infarction

Cardiol J 2009, 16:317-320.

38 Dickneite G, Pragst I, Joch C, Bergman GE: Animal model and clinical

evidence indicating low thrombogenic potential of fibrinogen

concentrate (Haemocomplettan P) Blood Coagul Fibrinolysis 2009 in

press.

39 Dart AB, Mutter TC, Ruth CA, Taback SP: Hydroxyethyl starch (HES) versus

other fluid therapies: effects on kidney function Cochrane Database

Syst Rev 2010:CCD007594.

doi: 10.1186/cc8960

Cite this article as: Grottke et al., Effects of different fibrinogen

concentra-tions on blood losss and coagulation parameters in a pig model of

coagul-opathy with blunt liver injury Critical Care 2010, 14:R62